Rheumatoid arthritis, like several other autoimmune diseases, is strongly associated with particular HLA alleles. However, the underlying immunological mechanisms which relate specific HLA susceptibility genes to disease pathogenesis are poorly understood. Structural studies have greatly clarified the details of the trimolecular complex, in which polymorphic sites on the HLA molecules bind peptide and TCR residues, providing the specific restriction properties which promote interaction. Relating these structural details to function is challenging, since at least three key immunological parameters-T cell selection, T cell regulation, and T cell activation-can be strongly influenced by subtle alterations in the trimolecular interaction. In this project, we propose to test the disease-promoting relationships of specific RA-associated HLA class II alleles through analysis of each of these three immunological parameters. Specifically, the avidity and TCR bias of T cell recognition of RA-associated class II molecules during thymic selection will be studied using class II tetramer binding in concert with a novel heterozygous murine model expressing two closely-related disease-associated DR4 alleles. Maturation and function of Treg populations will be followed in this model system, using markers which directly compare Treg commitment with TCR-ligand avidity. The influence of HLA risk alleles on lowering thresholds for peripheral activation will be evaluated in an evaluation of synergistic interactions between the RA-associated DR*0401 and *0404 molecules. Parallel analysis of human in vitro T cell responses using the same tetramer reagents will be used to validate the murine model studies. This work integrates studies of HLA-disease associations with current immunological concepts of T cell biology in an attempt to advance our understanding of mechanisms of human autoimmune disease.